Antibiotics have been effective in treating infectious diseases,
but resistance to these drugs has led to the emergence of new and the
reemergence of old infectious diseases, One strategy employed to
overcome these resistance mechanisms is the use of combination of drugs,
such as [beta]-lactams together with [beta]-lactamase inhibitors.
Several plant extracts have exhibited synergistic activity against
microorganisms. This review describes in detail, the observed synergy
and mechanism of action between natural products including flavonoids and essential oils and synthetic drugs in effectively combating
bacterial, fungal and mycobacterial infections. The mode of action of
combination differs significantly than that of the same drugs acting
individually; hence isolating a single component may lose its importance
thereby simplifying the task of pharma industries.

Infectious diseases are caused by bacteria, viruses, parasites and
fungi, and it is due to a complex interaction between the pathogen, host
and the environment. The discovery of antibiotics had eradicated the
infections that once ravaged the humankind. But their indiscriminate use
has led to the development of multidrug-resistant pathogens. Around
90-95% of Staphylococcus aureus strains worldwide are resistant to
penicillin (Casal et al., 2005) and in most of the Asian countries
70-80% of the same strains are methicillin resistant (Chambers, 2001).
There are considerable reports on the progress of resistance to the last
line of antibiotic defense, which has led to the search for reliable
methods to control vancomycin-resistant Enter-ococci (VRE) and S. aureus
(VRSA), and methicillin-resistant S. aureus (MRSA). In addition, the
synergy between tuberculosis and the AIDS epidemic, along with the surge
of multidrug-resistant isolates of Mycobacterium tuberculosis, has
reaffirmed it as a primary health threat. Multidrug-resistant TB (MDRTB)
is associated with high death rates (50-80%), spanning within a
relatively short period of time (4-16 weeks) from diagnosis to death
(WHO, 2004). In developing countries, MDRTB has increased in incidence
and it interferes with TB control programs.

Plant-derived antibacterials are always a source of novel
therapeutics. A quick look at the way nature, especially plants, are
tackling the issue of infection will provide a deeper understanding of
the methodology, which needs to be adopted for the design and
development of novel highly effective antiinfectious agents in general,
and antimycobacterials in particular. The scarcity of infective diseases
in wild plants is in itself an indication of the successful defense
mechanisms developed by them. Plants are known to produce an enormous
variety of small-molecule (MW < 500) antibiotics--generally
classified as 'phytoalexins'. Their structural space is
diverse having terpenoids, glycosteroids, flavonoids and polyphenols. Be
that as it may, it is interesting to note that most of these small
molecules have weak antibiotic activity--several orders of magnitudes
less than that of common antibiotics produced by bacteria and fungi. In
spite of the fact that plant-derived antibacterials are less potent,
plants fight infections successfully. Hence, it becomes apparent that
plants adopt a different paradigm--"synergy"--to combat
infections. A case in study to reiterate this view is the observation on
the combined action of berberine and 5'-methoxyhydnocarpin, both of
which are produced by berberry plants. Berberine, a hydrophobic alkaloid
that intercalates into DNA, is ineffective as an antibacterial because
it is readily extruded by pathogen--encoded multidrug resistance pumps
(MDRs). Hence, the plant produces 5'-methoxyhydnocarpin that blocks
the MDR pump (Stermitz et al., 2000). This combination is a potent
antibacterial agent (Lewis and Ausubel, 2006). Using this cue, Ball et
al. (2006) reported that covalently linking berberine to IN[F.sub.55],
an inhibitor of MDR, results in a highly effective antibiotic that
readily accumulates in bacteria.

This paper introduces and provides examples of synergistic
interactions of the secondary metabolites of plants with antibiotics in
the treatment of infectious diseases. The understanding of the molecular
mechanisms of synergy would pave a new strategy for the treatment of
infectious diseases, overcome drug-resistant pathogens, and decrease the
use of antibiotics and hence the side effects created by them.

Synergy towards bacterial infection

The development of antibiotic resistance can be natural (intrinsic)
or acquired and this can be transmitted within same or different species
of bacteria. Natural resistance is achieved by spontaneous gene mutation
and the acquired resistance is through the transfer of DNA fragments
like transposons from one bacterium to another. Bacteria gains
antibiotic resistance due to three reasons namely: (i) modification of
active site of the target resulting in reduction in the efficiency of
binding of the drug, (ii) direct destruction or modification of the
antibiotic by enzymes produced by the organism or, (iii) efflux of
antibiotic from the cell (Sheldon, 2005). One strategy employed to
overcome these resistance mechanisms is the use of combination of drugs.
Inhibitors of [beta]-lactamases have been long known and they are
administered with antibiotics as co-drugs. The most successful strategy
that has been adopted to overcome the resistance to penicillinase is by
administering clavulinic acid, with the drugs sulbactam and tazobactam
(Lee et al., 2003b). But the frequent use of clavulanate has led to the
emergence of resistant bacterial strains (Blasquez et al., 1993; Enright
et al., 2002). The appearance of extended spectrum [beta]-lactamase and
resistance against IMP-1 (a new [beta]-lactam), cephalosporins and
carbapenems have further necessitated the need for developing new
[beta]-lactamase inhibitors (Chaibi et al., 1999).

[FIGURE 1 OMITTED]

The secondary metabolites from plant are good sources for
combination therapy. As shown in Fig. 1, there are a wide range of
phytochemicals which act as multidrug resistance modifiers depicted and
their mechanism of action is discussed in the following sections.

Receptor or active site modification

For selective antimicrobial action the target site plays a vital
role. Introduction of mutations in the target site alters it, leading to
a reduction in the activity of the drug towards the microbe. Two
examples of receptor (target) modification are (a) mutations in RNA
polymerase and DNA gyrase, rendering rifamycins and quinolones inactive
(Heep et al., 2000; Willmott and Maxwell, 1993), and (b) modification in
the structural confirmation of penicillin-binding proteins (PBPs)
resulting in the development of penicillin resistance. The most
important example of a target change is the production of PBP2a, an
altered transpeptidase.

[beta]-Lactam antibiotics (BLA) are highly specific inhibitors of
the metabolism of peptidoglycan and they target the membrane bound
D,D-peptidase domain of the PBPs (Ghysen, 1994). These peptidases cross
link the bacterial peptidoglycan cell wall which maintain the integrity
of the latter (Berger-Bachi and Rohrer, 2002). S. aureus acquires
resistance to all penicillins and cephalosporins with the acquisition of
the gene mecA (Enright et al., 2002), which is carried on a large
genetic element called the staphylococcal cassette chromosome mec (SCC mec). This is acquired by a parasexual horizontal transfer from a
coagulase-negative Staphylococcus sp. (Ito et al., 2003). The house
keeping PBPs are BLA sensitive whereas the MecA (PBP 2a) have reduced
affinity to BLA (Lu et al., 1999). A number of BLAs, including modified
cephalosporins (Vouillamoz et al., 2004), carbapenems (Kurazono et al.,
2004) and trinem (Ferrari et al., 2003) have been designed with enhanced
activity against PBP2a.

Another approach to overcome resistance is to include inhibitors of
the PBP2a in the treatment strategy. A number of reports are available
listing the synergistic interactions of BLA with natural compounds to
overcome resistant microorganisms. The later includes catechins
(Camellia sinesis) (Takahashi et al., 1995), EGCg (Epigallocatechin
gallate) from green tea (Suresh et al., 1997), tellimagrandin I and
rugosin B from rose red (Rosa canina) (Shiota et al., 2000), baicalin
from Scutellaria amoena (Liu et al., 2000) and corilagin from
Arctostaphylos uva-ursi (Shimizu et al., 2001). Corilagin, a polyphenol from Arctostaphylos uva-ursi is found to markedly reduce the Minimum
Inhibitory Concentration (MIC) of [beta]-lactams in MRSA. Shimizu et al.
(2001) suggest that there are two possibilities regarding the mechanism
of action of corilagin, namely inhibition of PBP2a activity or
inhibition of its production. They later reported that the PBP2a of MRSA
cells grown in the presence of corilagin or tellimagrandin I lost its
ability to bind to BOCILLIN FL, a fluorescent-labeled benzylpenicillin (Shiota et al., 2004).

Studies through reverse transcription-PCR and a semiquantitative
PBP2a latex agglutination assays indicted that, EGCg did not suppress
either the mRNA expression of PBP2a or its production. But the synergy
between EGCg and BLA was achieved since both directly or indirectly
attacked the same target site namely, peptidoglycan present on the cell
wall (Yam et al., 1998; Zhao et al., 2001). EGCg was found to
synergistically enhance the activity of carbapenems against MRSA but the
mechanism of action has not been studied (Hu et al., 2002). Nicolson et
al. (1999) have shown that diterpene derivative 416 potentiated the
activity of methicillin by significantly reducing the expression of
PBP2a.

There is a wide list of phytochemicals which act as inhibitors and
a few of them are glycosylated flavones suppressing topoisomerase IV
activity (Bernard et al., 1997), myricetin inhibiting DnaB helicase
(Griep et al., 2007), allicin inhibiting RNA synthesis (Feldberg et al.,
1988) and compounds from the plant Polygonum cuspidatum inhibiting
bacterial DNA primase (Hegde et al., 2004). These phytochemicals when
used in combination with other classes of antibiotics have the potential
to either inhibit the modified targets or exhibit a synergy by blocking
one or more of the other targets in the metabolic pathway. Table 1 lists
the synergy observed between natural products and commercial antibiotics
against bacteria.

Bacterial cells spend a considerable amount of energy to resist
antibiotics. One way the cells achieve active drug resistance is by the
synthesis of enzymes that selectively target and destroy or modify the
antibiotics. The various enzymatic strategies that lead to antibiotic
inactivation are through hydrolysis, group transfer or redox mechanisms
(Wright, 2005). Hydrolytically susceptible chemical bonds (such as ester
or amide bonds) are cleaved by enzymes that are expressed by the
resistant organisms. The modification of the active group in the drug
through acylation, phosphorylation, glycosylation, nucleotidylation or
ribosylation by the organism could make the former innocuous. Redox
mechanism involves the oxidation-reduction of the antibiotics leading to
the information of inactive compound (Wright, 2005).

[beta]-Lactamases are one such family of enzymes that cleave the
[beta]-lactam ring of cephalosporins and penicillins. They act through
the serine residue in the active site of the enzyme or through the
activation of the Z[n.sup.2]+center (Bush, 1998, 2002). inhibitors of
[beta]-lactamases have long been known. The combination of ampicillin and sulbactam inhibits [beta]-lactamase and increases the spectrum of
activity of the former. Zhao et al. (2002) have confirmed that EGCg
inhibits the penicillinase produced by S. aureus thereby restoring the
activity of penicillin. It acts in a dose-dependent manner, with 50%
inhibition at a concentration of 10[micro]g/ml. The combination of
ampicillin and sulbactam is effective but is not powerful enough against
MRSA and strains producing [beta]-lactamases. When they are further
combined with EGCg, the MI[C.sub.90] of this combination is reduced to
4mg/ml from an initial value of 16mg/ml (Hu et al., 2001). The potent
synergy between these concoctions could possibly have clinical use.

Reduced accumulation of the antibiotic within the bacterial cell

Reduced accumulation of the antibiotic inside the microorganism could be because of two reasons namely decreased permeability of the
drug through the outer membrane of the cell or, the efflux of the
accumulated drug out of the cell.

Decreased outer membrane permeability

Cells of Gram-negative bacteria are surrounded by an additional
membrane (outer membrane, OM), which provide them with a hydrophilic surface and functions as a permeability barrier for many external
hydrophobic agents including detergents, hydrophobic dyes and
antibiotics (Helander et al., 1997a; Vaara, 1992, 1999; Nikaido and
Vaara, 1985). This barrier is due to the presence of lipopolysaccharide (LPS) molecules in the outer leaflet (Nikaido, 2003; Nikaido and Vaara,
1985), which makes up to 75% of the total membrane surface and forms
specific contacts with integral outer membrane proteins (Omp), such as
porins (Alexander and Rietschel, 2001; Bos and Tommassen, 2004).
Bacterial lipoproteins anchor the OM to the periplasmic peptidoglycan
layer (Brade et al., 1999). Divalent cations are tightly associated with
the anionic membrane-proximal regions of the LPS molecules,
strengthening the structure (Vaara, 1992). Some Gram-negative bacteria
are known to contain glycosphingolipids instead of LPS in their OM
(Kawahara et al., 1991).

Bivalent cations contribute to the stability of the OM by creating
electrostatic interactions between the proteins and LPS (Leive, 1965;
Vaara, 1981, 1999). EDTA is a chelator which sequesters these ions.
Treatment with EDTA releases a large proportion of LPS from the OM,
exposing the phosholipids and creating a hydrophobic pathway (Leive,
1965). EDTA has been reported to potentiate the activity of cell wall
degrading agents including lysozyme, nisin and biocides (Leive, 1965;
Vaara, 1981; Walsh et al., 2003a, b). In addition, there are a wide
range of permeabilizers such as polycationic polymyxin B nonapeptide,
which interact with and disorganize the anionic LPS thereby sensitizing
the bacteria to hydrophobic antibiotics (Vaara and Vaara, 1983a, b).
Essential oils such as thymol and carvacrol as membrane permeabilizers
(Fig. 1) have been studied by Helander and co-workers (Helander et al.,
1998). Magnesium chloride disrupts the activity of EDTA and
polyethylenimine, but it has no effect on the activity of carvacrol or
thymol. This indicates that essential oils neither chelate nor
intercalate with LPS by replacing the divalent cations which stabilize
the OM (Helander et al., 1997a; Vaara, 1992).

Active efflux

The development of multidrug resistance pumps (MDRs) is one of the
defense mechanisms employed by bacteria against the accumulation of
antimicrobial drugs inside the cell. These efflux pumps either use ATP
hydrolysis or ion gradient to expel the antibiotics. They are grouped
into five major classes namely, the adenosine triphosphate (ATP)-binding
cassette (ABC) superfamily (Veen and Konings, 1998; Veen et al., 1996),
the major facilitator superfamily (MFS) (Pao et al., 1998), the small
multidrug resistance family (SMR) (Paulsen et al., 1996), the
resistance-nodualtion-cell division RND superfamily (Saier et al., 1994)
and the multidrug and toxic compound extrusion (MATE) family (Brown et
al., 1999). Of these the RND, SMR and MATE classes are unique to
prokaryotes (Lynch, 2006).

The efflux pumps of S. aureus Qac A (MFS family), Smr (SMR family)
and Nor A (MFS family) have been well characterized. The Nor A efflux
pump is responsible for fluoroquinolone resistance (Yoshida et al.,
1990); Qac A is responsible for acriflavine and ethidium bromide
resistance (Littlejohn et al., 1992) and Tet (K) and Msr (A)
transporters are specific to tetracycline and macrolide efflux (Renau et
al., 1999). Renau et al. (1999) have developed the first broad-spectrum
RND pump-inhibitor, M[C.sub.-207,110]
(phenylalanyl-arginyl-[beta]-naphthy-lamide), which potentiates the
activity of levofloxacin, particularly the RND pumps against wild-type
P. aeruginosa. Although these compounds are not effective antimicrobial
agents by themselves, they reverse the resistance by blocking the efflux
pumps.

Secondary metabolites of plants have shown to possess considerable
activity against Gram-positive bacteria but not against Gram-negative
species or yeast. In Gram-negative species, the outer membrane is a
fairly effective barrier for amphiphatic compounds (Lewis and
Lomovskaya, 2001). A set of multidrug resistance pumps (MDRs) extrude
amphiphatic toxins across the outer membrane (Lewis, 2001; Nikaido,
1999). Tegos et al. (2002) have shown that MDR inhibitors
M[C.sub.207,110] and IN[F.sub.271] dramatically increase the
effectiveness of a set of 11 plant antimicrobials (e.g. Rhein,
resveratrol, gossypol, berberine) against Gram-negative bacteria. By
contrast, certain plant-derived natural products can modulate MDR. For
example carnosic acid (from Rosmarinus officinalis) (Oluwatuyi et al.,
2004), and a penta substituted pyridine (from Jatropha elliptica)
(Marquez et al., 2005), act as inhibitor of the Nor A efflux pump and
restore the level of intracellular drug concentration.

Two isopirmarane diterpenes from the Lycopus europaeus enhance the
activities of tetracycline and erythromycin against two strains of S.
aureus. Otherwise these strains are highly resistant to these
antibiotics due to the presence of multidrug efflux pumps, Tet (K) and
Msr (A) (Gibbons et al., 2003). EGCg increases the accumulation of
tetracycline in S. aureus strains by inhibiting the Tet (K) and Tet (B)
efflux pumps (Roccaro et al., 2004). EGCg also enhances the activity of
norfloxacin against a Nor A harboring S. aureus strain (Gibbons et al.,
2004). Isoflavones isolated from Lupinus argenteus act in synergy with
norfloxacin against a mutant of S. aureus by inhibiting the MDR pump.
Reserpine, a plant alkaloid potentiates the activity of fluoroquinolones
(Schmitz et al., 1998) and tetracycline against multidrug-resistant S.
aureus strain (Gibbons and Udo, 2000). Reserpine has been shown to
inhibit LmrA, the MDR ABC efflux system of L. lactis (Marquez et al.,
2005), but unfortunately bacterial resistance to this natural product
has been observed (Ahmed et al., 1993). The calcium channel antagonist
verapamil, another known inhibitor of P-gp, also inhibits several
bacterial ABC efflux pumps, including LmrA (Lee et al., 2003a; Pasca et
al., 2004; Choudhuri et al., 2002).The efflux pump inhibitors from
natural sources discussed so far can be co-administered with the
antibiotic to decrease the degree of resistance of the bacteria to the
drugs, reverse the acquired resistance of the microorganism or reduce
the emergence of resistant bacterial strains (Marquez et al. 2005).

Synergy and MDRTB therapy

Tuberculosis has established itself as a primary health threat. Few
new agents are in development today for treating TB, and none has been
designed specifically to shorten the treatment regimen and provide the
break-through in therapy that is sorely needed if the epidemic is to be
brought under control. Drug design targeting the latency stage and
synergistic interaction between the various drug candidates might prove
to be good alternatives.

Antimycobacterial treatment has always been a combination therapy.
Today's TB treatment, which dates back to 1970s, is long and
burdensome, requiring at least 6 months of multidrug chemotherapy. Novel
targets are being identified alongside developing better drugs for known
targets. Synergistic interaction between these drug like molecules is
also gaining sufficient attention from the researchers (Chen et al.,
2006; Vinogradova et al., 1999). Combination studies with natural
products from plants and synthetic drugs are limited to few reports.
Totarol ferulenol and plumbagin were observed to increase the potency of
isonicotinic acid hydrazide by fourfold against Mycobacterium sp. (Mossa
et al., 2004). A napthoquinone 7-methyljuglone, isolated from the roots
of Euclea natalensis in combination with isoniazid or rifampicin resulted in a four-to sixfold reduction in the MIC of the synthetic
drugs (Bapela et al., 2006). An aqueous extract from Cuminum cyminum
seeds produced a 35% enhancement of rifampicin levels in rat plasma.
This activity was due to a flavonoid glycoside, 3',
5-dihydroxyflavone-7-0-[beta]-D-galacturonide
4'-0-[beta]-D-glucopyranoside, found in the natural product. The
altered bioavailability profile of rifampicin could be attributed to the
permeation enhancing effect of this glycoside (Sachin et al., 2007).

Antifungal agents and synergism

Fungi have higher number of chromosomes and complex nuclear
membrane, cell organelles and cell wall composition. Since the last
three decades, the rate of death every year due to fungal infections has
risen significantly. With the increased use of antifungal agents there
is an increase in the number and variety of fungal strains resistant to
these drugs. Also the present antifungal therapeutics is often toxic.
Alternative therapy needs to be developed to suppress the emergence of
antifungal resistance. This can be achieved by the use of combinations
of existing agents or the development of new, safer and effective agents
primarily from plant sources which can exhibit synergy with drugs. Table
2 lists the reported synergy observed between natural products and drugs
towards fungal species.

In a recent study by Han (2007), a synergistic effect of grape seed
extract (GSE) with amphotericin B was observed in both in vitro and in
murine model of disseminated candidiasis due to Candida albicans. Mice
treated with combination of amphotericin B and GSE or amphotericin B
alone survived 62.4 and 38.4 days, respectively. The combination therapy
reduced more than 75% of amphotericin B required to achieve the same
level of inhibition.

In vitro evaluation of synergy

The accurate prediction of synergy between commercial drugs or
between a drug and a natural product based upon the results of in vitro
testing is very crucial. A number of methods are used to detect synergy.
However, the checkerboard and time-kill curve methods are the two most
widely used techniques and the former is a relatively easy test to
perform (White et al., 1996). The checkerboard is prepared in microtiter
plate for multiple combinations of two antimicrobial agents in
concentrations equal to, above, and below their minimal inhibitory
concentrations for the microorganism that is being tested. Each row (x
axis) in the plate will contain the same diluted concentration of the
first antimicrobial compound; while the concentration in each subsequent
row will be half this value. Similarly each column (y axis) in the plate
will contain the same diluted concentration in each subsequent column
will be half this value. The drugs combination in which the growth is
completely inhibited is taken as effective MIC for the combination.

The time-kill method assesses the bactericidal activity of the
individual as well as different concentration of the combination of
drugs as a function of time. It is a labor intensive and time-consuming
process (White et al., 1996). Tubes containing individual compounds and
combination of compounds with concentrations ranging from one-quarter to
twice the MIC for the bacterial strain of interest (NCCLS, 1987) are
prepared. The tubes are inoculated with about 5 X [10 sup.5] colony
forming units/ml of the strain, and they are incubated overnight.
Aliquots of the samples from Oh of incubation (reflecting the initial
inoculum) and 24 h of incubation (reflecting exposure of bacteria to the
compound) are plated onto agar plates. Synergy is defined as a 100-fold
or greater decrease in colony count at 24 h by the combination of agents
with reference to the starting inoculum and also when compared to the
most active single agent (Saiman, 2007).

E test is another method of recent origin. It consists of two
plastic strips coated with a continuous gradient of each of the compound
on one side. For evaluation of synergy, one compound strip is placed
onto an agar plate for 1 h and then removed, and the second compound
strip is placed on top of the gradient left behind by the first. The MIC
of the combination is taken as the value at which the two inhibition
zones intersect. If the use of the E strip could be standardized for
testing the synergy of drugs and the results obtained could be
demonstrated to be similar to those determined by established methods,
this new test method would represent an attractive alternative to the
labor-intensive procedures. Further, this method could be performed on a
routine basis in a clinical microbiology laboratory (White et al.,
1996). The standardization of these techniques for routine routine
laboratory testing is the need because of the common use of combination
therapies against the growing numbers of multiple drug-resistant
strains.

Analysis of the synergy data

In all the above methods the interaction between the two
antimicrobial agents is estimated by calculating the fractional
inhibitory concentration of the combination (FIC) index. The FIC of each
drug is calculated by dividing the concentration of the compound present
in that well in combination where complete inhibition of growth of the
microorganism is observed by the MIC of that compound alone to inhibit
the microorganism. The FIC of the combination is then the sum of these
two individual FIC values. When the FIC index of the combination is
equal to or less than 0.5, the combination is termed as synergistic;
when FIC index falls between 0.5 and 4.0, it indicates 'no
interaction' between the agents, and a value above four indicates
antagonism between the two compounds (Odds, 2003).

A convenient graphical way of representing the results of
combination studies is by the use of an 'isobologram',
introduced by Loewe and Muischnek (1926). It is independent of the
mechanism of action, makes no assumption about the behavior of each
compound. So it is applicable to multiple component mixtures.
Combination of drugs X and Y that shows inhibition of the growth of the
organism are represented in a graph using rectangular coordinates as
(x,y) for the respective doses. In this format, the dose of drug X alone
as (a) and drug Y alone as (b) are represented along the axes as (a,0)
and (0,b). The straight line connecting these points is called the
'line of additivity'. This line provides a convenient means
for visually discriminating additive from non-additive interactions on
the basis of whether or not the coordinate of the combination falls on
(additive), below (superadditive) or above (subadditive) this line. The
determination requires statistical evaluation (Tallarida et al., 1989)
because the technique obtains the individual or combination of doses as
random variables from the dose response data and there is always an
error involved in the estimation (Tallarida and Raffa, 1996). If synergy
is occurring, the dose of the combination needed to produce the same
effect will be less than the sum of the individual components and then
the curve will be concave. In antagonism, the dose of combination will
be greater than expected and the curve will be convex. The
'isobole' method has been well explained with several examples
by Williamson (2001).

Synergistic interactions in other therapies

The successful use of combinations of plant extracts is not only
observed in antiinfective therapy, but also seen in the treatment of
several disorders including cancer, HIV, inflammatory, stress-induced
insomnia, osteoarthritis and hypertension (Williamson, 2001).
Conventional medicine applies the "silver bullet" method,
where single target therapy is employed. The recent trend has been the
"herbal shotgun" method like Ayurveda, where multitargeted
approach of the herbals and drugs is used. Today illness such as cancer,
AIDS, hypertension, etc., are successfully treated with combination of
3-5 synthetic drugs. Cannabis extract is found to act in synergy as
antispastic agent in mice than tetrahydrocannabinol at an equivalent
dose (Baker et al., 2000; cit. at Wagner, 2006). Ginkgolide A and B has
been seen to act in synergy in the inhibition of PAF- induced
thrombocyte aggregation (Wagner, 2001, 2006) In the case of cancer
chemotherapy, the molecular targets for the phytochemicals is diverse
hence it necessitates the need to understand the degree of its
interaction with synthetic drugs. Multitargeted therapy approach
involving the application of phytochemicals or phytoextracts and
synthetic drugs as anticancer agents has been detailed in a review
(Hemaiswarya and Doble, 2006).

Conclusions

Before prescribing an antibiotic treatment the guidelines usually
suggest that a specimen containing the suspected organism is sent for
culture and sensitivity. Microbiology departments, for their part, use
in vitro sensitivity of isolates taken from patients with bacterial
infections to recommend which antibiotic(s) to prescribe or to use as an
empirical guide for treatment in other situations. There are a number of
reports available on the different antibiotic combinations tested in
vitro and applied to clinical scenario. But there are no reports on the
use of natural products and synthetic drug combinations used in the
clinical settings. As discussed in the review, there is plenty of hope
for the purified natural products to be used in combination with
antibiotics as antiinfective drugs. EGCg, demonstrates a synergistic
behavior with antibiotics by destroying the [beta]-lactamase activity as
well as by acting on the peptidoglycan of the cell wall. The safe
consumption of tea for thousands of years indicates its low toxicity.
EGCg, the principal constituent of tea, is absorbed through the
digestive tract and distributed to many organs in animals and humans.
This indicates the high bioavailability of EGCg which could enhance the
activity of antibiotic under in vivo conditions. Thus the undesirable
side effects of antibiotics on human and animal health could be possibly
reduced by replacing at least in part the synthetic substances by
negligibly toxic, highly specific antimicrobial compounds.

Several reports are available that describe the action of secondary
metabolites from plants as antimicrobial agents. While the screening of
natural compounds for antimicrobial activity is by itself a research
area of major significance, the development of compounds with resistance
modifying action is of interest since currently there are no known
agents presently in use in clinics. In order to select a compound that
could act in synergism with a drug it is necessary to understand the
complete molecular mechanism of the drug action in the presence and
absence of the natural compound. The problems that still need to be
addressed are stability, selectivity and bioavailability of these
natural products, and any adverse herb-drug interaction. To overcome
multidrug resistance in the antimicrobial therapy a combination of drugs
has to be used. The maximum benefit can be achieved when the
pharmacokinetics of natural product and the antibiotic combination
match. This does not mean that pharmacokinetic profiles for both agents
should be identical. The optimal ratio and dosing regimens should be
explored for higher efficacy and decreased toxicological profiles.
Animal models with engineered strains lacking the particular resistant
genotype can be used to very precisely define the pharmacokinetic and
pharmacodynamic targets followed by regulated clinical trials. Even in
vitro screening procedures for drug combination are time-consuming
process which should be speeded up to achieve quick breakthroughs in
combination therapy. Techniques such as isobologram can be used
successfully to demonstrate regions of synergy between drug combinations
from other regimes.

The recent developments in genomics, proteomics and metabolomics
have created a new platform to distinguish the synergistic efficacy of
phytoextracts and for the determination of their mode of action. By the
application of the "-omic" technologies it should be possible
to detect the mechanism of action as the gene/ protein expression
profiles of the combination of drugs can be entirely different from the
ones induced by the single drugs. This may lead to new phyto-based
paradigms towards the use of complex plant mixtures in medicine
(Ulrich-Merzenich et al., 2007).

As seen from this review, the number of natural compounds acting in
synergy with synthetic drugs towards fungal and Mycobacterium species
are minimal. This could be due to limited understanding of the mechanism
of action of drugs against these organisms or insufficient screening of
natural compounds. So research should be focused towards this direction
to identify more natural compounds which exhibit synergistic behavior.